Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents

Six crude oil-degrading bacterial strains isolated from different soil and water environments were combined to create a defined consortium for use in standardized efficacy testing of commercial oil spill bioremediation agents (OSBA). The isolates were cryopreserved in individual aliquots at pre-determined cell densities, stored at −70°C, and thawed for use as standardized inocula as needed. Aliquots were prepared with precision (typically within 10% of the mean) ensuring reproducible inoculation. Five of the six strains displayed no appreciable loss of viability during cryopreservation exceeding 2.5 years, and five isolates demonstrated stable hydrocarbon-degrading phenotypes during inoculum preparation and storage. When resuscitated, the defined consortium reproducibly biodegraded Alberta Sweet Mixed Blend crude oil (typically ± 7% of the mean of triplicate cultures), as determined by quantitative gas chromatography–mass spectrometry of various analyte classes. Reproducible biodegradation was observed within a batch of inoculum in trials spanning 2.5 years, and among three batches of inoculum prepared more than 2 years apart. Biodegradation was comparable after incubation for 28 days at 10°C or 14 days at 22°C, illustrating the temperature tolerance of the bacterial consortium. The results support the use of the synthetic consortium as a reproducible, predictable inoculum to achieve standardized efficacy tests for evaluating commercial OSBA. Received 31 August 1998/ Accepted in revised form 30 November 1998     

Regulation of antibiotic production by iron and oxygen during defined medium fermentations of Streptomyces clavuligerus

Summary When grown in a chemically defined medium, Streptomyces clavuligerus excreted cephamycin C, in addition to other components, throughout most of the growth phase. Ferrous iron and oxygen are required for the biosynthesis of this antibiotic and the concentration of these cofactors was manipulated to maximize cephamycin C production. The iron content of the chemically defined medium was shown to be sub-optimal for antibiotic production and the addition of 130 g/ml ferrous iron almost doubled the cephamycin C levels to 200 g/ml. When dissolved oxygen was maintained at saturation levels, only 60–80 g/ml cephamycin C was produced, and the intermediate penicillin N accumulated to high levels (50 g/ml). This suggests that the high concentration of dissolved oxygen had a greater effect on the enzymes catalysing the conversion of penicillin N to cephamycin C, than on those involved in the earlier steps of the pathway leading to the formation of penicillin N.     

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.     

Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.     

A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.     

Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.     

Changes in pattern of respiration and glucose utilisation in Candida utilis during the cell cycle: some variations with growth rate.

The release of 14CO2 from 14C-labelled glucose(G-1(-14)C, G-3,4(-14)C, G-6(-14)C) was followed in phased cultures of Candida utilis grown in a glucose- mineral salts medium under altered conditions of carbon:nitrogen limitation at doubling times of 2,4 and 6, h. Changes in oxygen uptake and CO2 evolution were observed and respirometric studies showed that the relative contributions of the Embden-Meyerhof-Parnas and hexose monophosphate pathways varied over the cell cycle and changed with growth rate. The results are discussed in relation to the growth metabolism of the cells.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.